Canine lyme disease vaccine

ABSTRACT

The present invention provides a vaccine for canine Lyme disease and methods of making and using the vaccine alone, or in combinations with other protective agents.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a non-provisional application that claims priorityunder 35 U.S.C. §119(e) of provisional application U.S. Ser. No.60/864,258 filed Nov. 3, 2006, the contents of which are herebyincorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to a vaccine for canine Lyme disease.Methods of making and using the vaccine alone or in combinations withother protective agents are also provided.

BACKGROUND

Canine Lyme disease is caused by infection with Borrelia species (spp.)spirochetes, including primarily B. burgdorferi sensu stricto (ss) inthe United States and B. burgdorferi ss, B. garinii, and B. afzelii inEurope (Baranton et al., Int. J. Sys. Bacteriol. 1992, 42:378-383;Hovius et al., J. Clin. Microbiol. 2000, 38:2611-2621). The spirochetesare transmitted as the infected Ixodes spp. ticks obtain a blood meal,and the resulting infection in canines results in clinical signs rangingfrom subclinical synovitis to acute arthritis and arthralgia (Jacobsonet al., Semin. Vet Med. Surg. 1996, 11:172-182; Summers et al, J. Comp.Path. 2005, 133:1-13). Importantly, the incidence of canine Lyme diseasecases continues to increase annually coincident with increased numbersof human cases (Haninkova et al., Emerg. Infect. Dis. 2006, 12:604-610).

The antibodies produced in response to infection with Borrelia spp. havetwo distinct functions, but heretofore, both responses could beineffective at eliminating sequestered spirochetes from a mammalianhost. A number of explanations have been postulated for this defect inthe normal immune response to natural infection, including antigenicvariation (Schwan, Biochem. Soc. Trans. 2003, 31:108-112; Tokarz et al.,Infect. Immun. 2004, 72:5419-5432) host mimicry (Barbour et al.Microbiol. Rev. 1986, 50:38-400), and intracellular localization (Ma etal., Infect. Immun. 1991, 59:671-678).

The most common humoral immune response is the production ofnon-specific binding/opsonizing (coating) antibodies that “mark” thespirochete for ingestion by phagocytic cells. Unfortunately, opsonizingantibodies are induced by several proteins common to othermicroorganisms (viz. 41 kDa proteins that comprise bacterial flagella),making their value for vaccination-induced antibody-mediated immunity,at best, questionable.

A second common immune response is the production of borreliacidal(lethal) antibodies. In contrast to opsonizing antibodies, borreliacidalantibodies recognize epitopes on only a few Borrelia spp. proteins.After binding to the specific target on the spirochete, theborreliacidal antibodies most commonly induce complement to form amembrane attack complex that kills the organism without the necessity ofscavenging by phagocytic cells.

The canine Lyme disease bacterins presently employed in vaccines weredeveloped to provide protection by inducing OspA borreliacidalantibodies (Hsien-Chu et al., JAVMA 1992, 201:403-411; Ma et al.,Vaccine 1996, 14:1366-1374; Wikle et al., Intern. J. Appt Res. Vet. Med.2006, 4:23-28; Straubinger et al., Vaccine 2001, 20:181-193) that killthe OspA-expressing spirochetes in the infected ticks as the parasitesprocure a bloodmeal (Fikrig et al., Proc. Natl. Acad. Sci. USA 1992,89:5418-5421). Straubinger et al., (Vaccine 2002, 20:181-193) hasreported that a whole cell vaccine induced significantly higher titersof borreliacidal antibodies than a recombinant OspA. Although suchvaccines have been reasonably successful, vaccination failures have beenreported (Levy et al. JAVMA 1993, 202:1834-1838; Ma et al., Vaccine1996, 14:1366-1374; Schutzer et al., N. Engl. J. Med. 1997,337:794-795).

It is now understood that the OspA borreliacidal antibodies generatedoften fail to sterilize feeding ticks, because the antibodies onlyrecognize B. burgdorferi ss (Jobe et al., J. Clin. Microbiol. 1994,32:618-622; Lovrich et al., Infect. Immun. 1995, 63:2113-2119) that areexpressing OspA, and the ticks are commonly infected with B. burgdorferiss spirochetes that are not expressing OspA (Fikrig et al., Infect.Immun. 1995, 63:1658-1662; Ohnishi et al., Proc. Natl. Acad. Sci. 2001,98:670-675). In addition, the ticks are commonly also infected withother pathogenic Borrelia spp. including B. afzelli and B. garinii(Ornstein et al., J. Clin. Microbiol. 2001, 39:1294-298), while the OspAantibodies are genospecies specific (Lovrich et al., Infect. Immun.1995, 63:2113-2119). Moreover, the ‘window of opportunity’ forprotection by OspA borreliacidal antibodies is limited even when thespirochetes are susceptible, because the expression of OspA, whichmediates attachment to the tick midgut (Pal et al., J. Clin. Invest.2000, 106:561-569), is down-regulated shortly after the infected tickbegins feeding (Schwan et al., Proc. Natl. Aced. Sci. USA 1995,92:2909-2913).

B. burgdorferi ss OspC is another potential target for borreliacidalantibody-mediated immunity (Rousselle et al., J. Infect. Dis. 1998,178:733-741). This protein appears to have an epitope that isresponsible for inducing borreliacidal antibodies and is conserved amongthe pathogenic Borrelia spp. (Lovrich et al., Clin. Diagn. Lab. Immunol.2005, 12:746-751). Although the specific function of the OspC proteinremains unknown, it has been suggested that OspC expression is requiredfor infection of mammals, but not for infection of ticks (Grimm et al.2004, Proc. Natl. Acad. Sci. 101(9):3142-3147). In any event, Lymedisease spirochetes express OspC shortly after the tick begins feeding(Schwan et al., Proc. Natl. Acad. Sci. USA 1995, 92:2909-2913) and mustcontinue to express OspC in order to establish an infection in mammals(Stewart et al., Infect. Immun. 2006, 74:3547-3553, Tilly et al.,Infect. Immun. 2006, 74:3554-3564). Therefore, the “window ofeffectiveness” of the OspC borreliacidal antibodies is increasedsignificantly compared to OspA borreliacidal antibodies.

It has been shown that the OspC protein can induce protectiveborreliacidal antibodies (Rousselle et al., J. Infect. Dis. 1998,178:733-741, Ikushima et al., FEMS Immunol. Med. Microbiol. 2000,29:15-21), but some previous “mapping” studies have localized theepitopes to highly heterogeneous regions of the protein (Buckles et al.,Clin. Vacc. Immunol. 2006, 13:1162-1165). Therefore, borreliacidal OspCantibodies raised against these regions would only provideantibody-mediated immunity against a small number of Borrelia spp.isolates. Lovrich et al., (Clin. Diagn. Lab. Immuno. 2005, 12:746-751)identified an OspC borreliacidal antibody epitope within the C-terminal7 amino acids (OspC7) of the protein. Most significantly, the epitope isconserved among the pathogenic Borrelia spp. However, traditionallaboratory B. burgdorferi ss isolates that express OspA (i.e., containthe ospA/ospB operon) cannot be manipulated in the laboratory to alsoinduce significant levels of OspC borreliacidal antibodies withoutsignificantly impairing their ability to induce OspA borreliacidalantibodies. Moreover, vaccinating with killed traditional laboratory B.burgdorferi ss isolates that express OspA does not induce borreliacidalOspC antibodies (Schwan et al., 1995, Proc. Natl. Acad. Sci. USA,92:2909-2913, Obonyo et al., 1999, J. Clin. Microbiol., 37:2137-2141).

Callister et al., (U.S. Pat. Nos. 6,210,676 and 6,464,985, incorporatedby reference herein) have suggested employing an immunogenic polypeptidefragment of OspC, alone or in combination with an OspA polypeptide, toprepare a vaccine to protect humans and other mammals against Lymedisease. Livey et al. (U.S. Pat. No. 6,872,550, incorporated byreference herein) also proposed a vaccine for immunizing against Lymedisease prepared from a combination of recombinant OspA, OspB, and OspCproteins. However, to date, no recombinant protein vaccine has beenshown to be an improvement over the vaccines that are currentlymarketed. Therefore, there remains a longstanding need in the art for animproved vaccine to protect mammals, and especially canines, from Lymedisease.

The citation of any reference herein should not be construed as anadmission that such reference is available as “prior art” to the instantapplication.

SUMMARY OF THE INVENTION

Accordingly, the present invention provides new immunogenic compositionsthat may be used in vaccines. In one aspect of the present invention, avaccine protects against Lyme disease. In a particular embodiment ofthis type, the recipient of the vaccine is a canine. In anotherembodiment, the recipient of the vaccine is a domestic cat. Otherdomestic mammals may be protected by the vaccines and/or methods of thepresent invention such as horses and/or cattle. The present inventionfurther provides combination vaccines for eliciting protective immunityagainst Lyme disease and other diseases, e.g., other canine infectiousdiseases. Methods of making and using the vaccines of the presentinvention are also provided.

A vaccine of the present invention comprises an immunologicallyeffective amount of organisms of a first or a single strain (optionallyinactivated) that expresses OspC antigen. The strain is one that, whengrown under standard culture conditions, is killed in the presence ofOspC-specific borreliacidal antibodies (complement is optionallyrequired), including borreliacidal antibodies against the conservedepitope OspC7, elicited in an animal vaccinated by B. burgdorferi ss50772 (ATCC No. PTA-439). In a particular embodiment of this type, thefirst or a single strain of Borrelia genospecies constitutivelyexpresses the OspC antigen. In a more particular embodiment, the firststrain is B. burgdorferi ss 50772 (ATCC No. PTA-439).

A vaccine composition of the present invention can further include animmunologically effective amount of inactivated organisms from one ormore additional strains (which may be collectively labeled herein as thesecond strain), from a pathogenic Borrelia genospecies. In a particularembodiment the second strain exhibits OspA and OspB antigens.

Examples of appropriate second strains include one or more of thefollowing: B. burgdorferi ss S-1-10 (ATCC No. PTA-1680), B. burgdorferiss B-31 (ATCC No. 35210), B. afzelii (e.g., available as ATCC No. 51567)and B. garinii (e.g., available as ATCC Nos. 51383 and 51991), B.burgdorferi ss DK7, B. burgdorferi ss 61BV3, B. burgdorferi ss ZS7, B.burgdorferi ss Pka, B. burgdorferi ss IP1, IP2, IP3, B. burgdorferi ssHII, B. burgdorferi ss P1F, B. burgdorferi ss Mil, B. burgdorferi ss20006, B. burgdorferi ss 212, B. burgdorferi ss ESP1, B. burgdorferi ssNe-56, B. burgdorferi ss Z136, B. burgdorferi ss ia, and/or anycombinations thereof.

The vaccine composition broadly includes from about 1×10⁴ to about1×10¹⁰ organisms per milliliter of each respective strain. In aparticular embodiment, the vaccine includes from about 1×10⁶ to about5×10⁹ organisms per milliliter of each respective strain. In anotherembodiment, the vaccine includes from about 1.0×10⁸ to about 5×10⁸organisms per milliliter of the (or each) second strain and from about5.0×10⁸ to about 5×10⁹ organisms per milliliter of the first strain.

The vaccine composition also can include a pharmaceutically acceptableadjuvant, e.g., such as, aluminum compounds (e.g., aluminum phosphate,aluminum hydroxide) metabolizable and non-metabolizable oils, blockpolymers, immune stimulating complexes, vitamins and minerals, andCARBOPOL® (e.g., CARBOPOL 941) In a particular embodiment thepharmaceutically acceptable adjuvant comprises a uniformly dispersedmicron size oil droplets in water emulsion (e.g., as sold under thetrademark Emulsigen®).

Optionally, the vaccine composition also includes a pharmaceuticallyacceptable immune stimulant, e.g., cytokines, growth factors,chemokines, supernatants from cell cultures of lymphocytes, monocyes, orcells from lymphoid organs, cell preparations and/or extracts fromplants, bacteria or parasites, or mitogens.

The invention further provides for a method of immunizing a canine, orother mammal, against pathogenic Borrelia spp., specifically B.burgdorferi ss, comprising injecting the canine with an immunologicallyeffective amount of the above described inventive vaccine. Such vaccinescan include from about 1×10⁸ to about 3×10⁹ organisms of each respectivestrain, for example. Vaccines may be administered by a route such as:intramuscular injection, subcutaneous injection, intravenous injection,intradermal injection, oral administration, intranasal administration,and combinations thereof. In a particular embodiment, after vaccination,the immunized canine produces borreliacidal antibodies.

The invention further provides serum obtained from a vaccinated animalthat contains borreliacidal antibodies that bind to B. burgdorferi ssOspC. Similarly, the invention provides for purified antibodies thatbind to OspC. In a particular embodiment, the serum contains asignificant proportion of OspC7-specific borreliacidal antibodies.

The invention further provides combination vaccines that include one ormore strains of the Borrelia genospecies of the present invention incombination with one or more other canine pathogens and/or immunogens,including, e.g., immunogens for eliciting immunity to canine distempervirus; canine adenovirus; canine parvovirus; canine parainfluenza virus;canine coronavirus; canine influenza virus; and/or Leptospira serovars,e.g., Leptospira kirschneri serovar grippotyphosa, Leptospirainterrogans serovar canicola, Leptospira interrogans serovaricterohaemorrhagiae, and/or Leptospira interrogans serovar pomona.Additional canine pathogens that can be added to a combination vaccineof the present invention include Leishmania organisms such as Leishmaniamajor and Leishmania infantum, Bordetella bronchiseptica; a Mycoplasmaspecies (e.g., Mycoplasma cynos); rabies virus; anaplasma organisms suchas anaplasma phagocytophilum and anaplasma platys; and Ehrlichia canis.

These and other aspects of the present invention will be betterappreciated by reference to the following Figures and the DetailedDescription.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a Western blot of a normal dog serum control (NS) or sera froma dog after vaccination (study day 43) with the test product (TP) orafter challenge (study day 134) with B. burgdorferi ss-infected ticks(NI). Note the presence of infection-specific antibodies against aprotein with a molecular weight of approximately 20 kDa.

FIG. 2A is a Western blot of a normal dog serum control (NS) or serafrom separate cohorts of individual dogs (numbered from 1-15) aftervaccination with placebo and challenge with B. burgdorferi ss-infectedticks (study day 134).

FIG. 2B is a Western blot of a normal dog serum control (NS) or serafrom separate cohorts of individual dogs (numbered from 1-15) aftervaccination with the test product. Note the presence of theinfection-specific 20 kDa antibodies in 14 (93%) dogs vaccinated withplacebo (FIG. 2A) and 0 (p<0.0001) dogs vaccinated with the test product(FIG. 2B).

FIG. 3A is a representative example of the histopathological changes inthe joints of placebo-vaccinated dogs that were infected with B.burgdorferi ss. The area within the rectangle represents a significantinfiltration of neutrophils and mononuclear cells characteristic ofcanine Lyme arthritis

FIG. 3B is a representative example of the absence of histopathologicalchanges in the joints of test product-vaccinated dogs that were infectedwith B. burgdorferi ss. The area within the oval is devoid of anyinfiltrating neutrophils and mononuclear cells.

FIG. 4 illustrates determination of OspC7 ELISA reactivity using normalsera (N) from individuals with no previous history (chart-review) ofLyme or Lyme-related symptoms (n=36), uncharacterized sera from blooddonors (BD, n=100) or individuals undergoing cholesterol screenings (CS,n=100), or sera from volunteers with blood factors or illnesses thatcommonly cross-react with B. burgdorferi ss antigens, includingantinuclear antibodies (ANA, n=20), rheumatoid factor (RF, n=20),mononucleosis (EBV, n=10), cytomegalovirus (CMV, n=10), syphilis (SYPH,n=13) or Rocky Mountain spotted fever (RMSF n=4). The solid line denotesthree standard deviations above the mean absorbance value of the normaland potential cross-reactive sera. Values above the line correspond topositive results to a 99% probability (1% background value).

FIG. 5 illustrates OspC7 ELISA reactivity using sera from probable Lymedisease patients with erythema migrans (n=86) representing patients withtypical Lyme lesions, likely Lyme patients with “atypical lesions”(n=22), and possible Lyme patients with constitutional symptoms (n=49)[earliest clinical signs]. The solid line denotes three standarddeviations above the mean absorbance value of the normal and potentialcross-reactive sera.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides vaccine compositions that include animmunologically effective amount of organisms from one or more strainsof a Borrelia genospecies that induce an effective borreliacidalantibody-mediated immunity in the recipient vaccinated animal. Whenorganisms of such strains are grown under standard Borrelia genospeciesgrowth conditions they are killed in the presence of OspC-specificantibodies, e.g., an antibody or antibodies elicited in an animalvaccinated with B. burgdorferi ss 50772 (ATCC No. PTA-439).

In one aspect of the present invention, the window of effectiveness ofthe antibodies induced by a vaccine of the present invention isincreased significantly compared to conventional vaccines based solelyon OspA borreliacidal antibodies. In another aspect, a vaccine of thepresent invention provides a better chance of stimulating a beneficialimmunologic memory response, e.g., due to the expression of OspC invivo, and/or an additional and/or enhanced protection against multiplepathogenic strains of Borrelia spp.

In one embodiment of the present invention, when the organisms of suchstrains are grown under standard Borrelia genospecies growth conditions,they constitutively express OspC antigen. In a particular embodiment ofthis type, when the organisms of such strains are grown under standardBorrelia genospecies growth conditions they are killed by a complementspecific reaction. In yet another embodiment, a significant proportionof the OspC-specific borreliacidal antibodies in the sera induced by avaccine comprising organisms of such strains are specific for theconserved epitope OspC7 and thereby, provide protection against multiplepathogenic Borrelia genospecies (e.g. B. burgdorferi ss, B. afzelii,and/or B. garinii). In still another embodiment, organisms of suchstrains exhibit no OspA or OspB antigen. In yet another embodiment,organisms of such strains have any two or more of these properties. Inyet another embodiment, organisms of such strains have any three or moreof these properties. In still another embodiment, organisms of suchstrains have any four or more of these properties. In a particularembodiment, organisms of such strains have all of these properties. Onesuch particular strain is B. burgdorferi ss 50772 (ATCC No. PTA-439).

In an alternative embodiment, the inventive vaccine is a compositionthat includes both a first strain as described above and an effectiveamount of organisms of at least a second strain. The second strain ispreferably from a pathogenic Borrelia genospecies that inducesborreliacidal OspA antibodies when administered as a part of theinventive vaccine. One or more additional compatible vaccine genospeciescan also be included therein. In addition, the vaccines of the presentinvention can include one or more other mammalian (e.g., canine)pathogens and/or immunogens.

In order to more fully appreciate the invention, the followingdefinitions are provided.

The use of singular terms for convenience in description is in no wayintended to be so limiting. Thus, for example, reference to acomposition comprising “a polypeptide” includes reference to one or moreof such polypeptides. In addition, reference to an “organism” includesreference to a plurality of such organisms, unless otherwise indicated.

As used herein the term “approximately” is used interchangeably with theterm “about” and signifies that a value is within fifty percent of theindicated value i.e., a composition containing “approximately” 1×10¹⁰organisms per milliliter contains from 5×10⁹ to 5×10¹⁰ organisms permilliliter.

The term “genospecies,” was first used and defined by G. Baranton etal., 1992, International J. of Systematic Bacteriology 42: 378-383, andis used herein in the same way that the term, “species” is employed indescribing the taxonomy of non-Borrelia organisms.

“Standard growth conditions” for culturing Borrelia genospecies requiregrowth at a temperature ranging from about 33° C. to about 35° C. in BSK(Barbour Stoenner Kelly) medium. BSK medium as described herein wasprepared according to Callister et al. [Detection of BorreliacidalAntibodies by Flow Cytometry, Sections 11.5.1-11.5.12, Current Protocolsin Cytometry, John Wiley and Sons, Inc. Supplement 26, (2003) herebyincorporated by reference herein in its entirety], (BSK medium is alsocommercially available, e.g., from Sigma, St. Louis, Mo.).

As used herein “OspC7” is an immunodominant OspC borreliacidal antibodyepitope located in a 7 amino acid region (Lovrich et al., 2005, Clin.Diagn. Lab. Immunol., 12:746-751, incorporated by reference herein inits entirety) within the C-terminal 50 amino acids of OspC, as disclosedby Callister et al. (U.S. Pat. Nos. 6,210,676 B1 and 6,464,985 B1, whichare incorporated by reference herein in their entireties) that isabsolutely conserved among the known pathogenic Borrelia spp. Thisconservation is readily confirmed by a BLAST search of the codon segmentencoding the 7 amino acid segment described by Lovrich et al. Such asearch, when conducted on Oct. 9, 2006 generated a results list of 100Borrelia species containing the above noted OspC 7-mer epitope codingsegment.

An “OspC-specific borreliacidal antibody” is one that is found, e.g., inthe serum of an animal vaccinated with B. burgdorferi ss 50772 (ATCC No.PTA-439), and is one that selectively binds to any epitope of the OspCantigen and kills the spirochetes dependent or independent ofcomplement. An “OspC7-specific borreliacidal antibody” is one that isfound, e.g., in the serum of an animal vaccinated with B. burgdorferi ss50772 (ATCC No. PTA-439), and is one that selectively binds to the 7C-terminal amino acids of OspC as described by Lovrich et al. [Clin.Diagn. Lab. Immunol., 12:746-751, (2005), incorporated by referenceherein in its entirety] and kills the spirochetes (generally by inducinga complement-mediated membrane attack complex). The specificity of OspCborreliacidal antibodies has been well-established. For example, OspCborreliacidal antibodies are detected commonly in Lyme disease sera bymeasuring the susceptibility of B. burgdorferi ss 50772 in aborreliacidal antibody test. Sera from human patients withclosely-related illnesses only rarely (2%) contain cross-reactiveantibodies that also kill strain 50772 (described in detail byCallister, et al., 1996, Clinical and Diagnostic Laboratory Immunology3(4): 399-402). Moreover, a peptide ELISA that uses the OspC7borreliacidal epitope accurately captures borreliacidal antibodies inLyme disease sera, and sera from patients with other closely relatedillnesses only rarely (<2%) contain cross-reactive antibodies that alsobind the OspC7 peptide (illustrated by FIGS. 4 and 5).

When a “significant proportion” of the OspC-specific borreliacidalantibodies in sera induced by a vaccine are specific for the conservedepitope OspC7, it means that there is a measurable reduction in theOspC-specific borreliacidal antibodies in the sera following theabsorption of that sera with OspC7. It is preferably defined as at leasta 2-fold reduction in the borreliacidal antibody titer of the seradetected by using B. burgdorferi ss 50772, and more preferably as a 2-to 4-fold, or greater reduction in the borreliacidal antibody titer ofthe sera following the absorption of that sera with OspC7.

A “complement specific reaction” is an antibody reaction that requiresserum complement to be present in order for Borrelia spp. organism(s) tobe killed by a borreliacidal antibody.

For the purposes of this invention, an “inactivated” Borreliaburgdorferi ss organism is an organism which is capable of eliciting animmune response in an animal, but is not capable of infecting theanimal. The Borrelia burgdorferi ss isolates may be inactivated by anagent selected from the group consisting of binary ethyleneimine,formalin, beta-propiolactone, thimerosal, or heat. In a particularembodiment, the Borrelia burgdorferi ss isolates are inactivated bybinary ethyleneimine.

The terms “adjuvant” and “immune stimulant” are used interchangeablyherein, and are defined as one or more substances that cause stimulationof the immune system. In this context, an adjuvant is used to enhance animmune response to one or more vaccine antigens/isolates. An adjuvantmay be administered to the target animal before, in combination with, orafter the administration of the vaccine. Adjuvants of the presentinvention may be obtained from any of a number of sources including fromnatural sources, recombinant sources, and/or be chemically synthesized,etc. Examples of chemical compounds used as adjuvants include, but arenot limited to aluminum compounds, metabolizable and non-metabolizableoils, block polymers, ISCOM's (immune stimulating complexes), vitaminsand minerals (including but not limited to: vitamin E, vitamin A,selenium, and vitamin B12), Quil A (saponins), and polymers of acrylicacid cross-linked with polyalkenyl ethers or divinyl glycol e.g.CARBOPOL®. Additional examples of adjuvants, that sometimes have beenreferred to specifically as immune stimulants, include bacterial andfungal cell wall components (e.g., lipopolysaccarides, lipoproteins,glycoproteins, muramylpeptides, beta-1,3/1,6-glucans) various complexcarbohydrates derived from plants (e.g., glycans, acemannan), variousproteins and peptides derived from animals (e.g., hormones, cytokines,co-stimulatory factors), and novel nucleic acids derived from virusesand other sources (e.g., double stranded RNA, CpG). In addition, anynumber of combinations of the aforementioned substances may provide anadjuvant effect, and therefore, can form an adjuvant of the presentinvention. One preferred adjuvant is Emulsigen®.

B. burgdorferi ss 50772 (ATCC No. PTA-439) as stated in U.S. Pat. No.6,210,676, and B. burgdorferi ss S-1-10 (ATCC No. PTA-1680) as stated inU.S. Pat. No. 6,316,005, were deposited with the American Type CultureCollection, 10801 University Boulevard Manassas (VA) 20110 on Jul. 30,1999, and Apr. 11, 2000, respectively. The co-owners of the rights tothe present invention individually hold the rights to these twoaforementioned patents.

It is also to be understood that this invention is not limited to theparticular configurations, process steps, and materials disclosed hereinas such configurations, process steps, and materials may vary somewhat.It is also to be understood that the terminology employed herein is usedfor the purpose of describing particular embodiments only and is notintended to be limiting, since the scope of the present invention willbe limited only by the appended claims and equivalents thereof.

Additional Vaccine Strains

Additional OspC Strains

In one aspect, the present invention provides the use of B. burgdorferiss 50772 (ATCC No. PTA-439) in a vaccine that protects against Lymedisease, either alone, or in combination with other Borrelia genospeciesstrains. Notably, B. burgdorferi ss 50772 was initially rejected as acandidate for being useful in a vaccine because it was incorrectlyreported that this strain did not express OspC (Anderson et al., 1996,J. Clin. Microbiol., 34:524-529).

The present invention further provides unique and specific criteria toselect/identify other such useful isolates for vaccines of the presentinvention. In particular, isolates can be selected/identified that aresusceptible to being killed by OspC-specific antibodies, and preferably,for also possessing one or more, or all of the following attributes: (i)the susceptibility to being killed by OspC-specific antibodies through acomplement specific reaction, (ii) the ability to induce OspCborreliacidal antibodies in a recipient vaccinated by that isolate;(iii) for a significant proportion of the OspC borreliacidal antibodiesso induced to be specific for the conserved epitope within theC-terminal 7 amino acids (OspC7); (iv) lacking the capacity to expressOspA and OspB; and/or (v) the ability to express OspC constitutively invitro.

A. Suppression of OspA/B Expression and Enhancement of OspC Expressionby Culture Conditions

It has been shown by Obonyo et al. (J. Clin. Microbiol. 1999,37:2137-2141) that OspA expression can be downregulated, and OspCexpression can be upregulated over a five day period by co-culturing anormal pathogenic Borrelia spp. strain (strains JMNT and N40 of B.burgdorferi ss) with a tick cell line (Ixodes scapularis ISE6). The mostpronounced effect was seen by culturing at 37° C. Thus, this method willconvert one or more conventional pathogenic Borrelia strains into a formuseful for a vaccine of the present invention.

B. Suppression of OspA/B Expression and Enhancement of OspC Expressionby Mutation

In a further embodiment, genetic manipulation of a Borrelia spp. strainthat originally expresses OspA and OspB is employed to downregulate orto delete the genes that code for the expression of OspA and OspB thatresults in the upregulation of OspC expression. Any art-known method ofgenetic manipulation is contemplated to be employed for this purpose.For example, an inactivated analog of the OspA/OspB gene is introducedinto a plurality of organisms of an available Borrelia spp, strain inthe form of a Borrelia spp compatible plasmid prepared with anantibiotic selection marker (see e.g., Grimm et al., 2004, Proc. Nat'lAcad. Sci 101(9):3142-3147 who report that OspC expression can beblocked in a Borrelia spp. by inserting OspC inactivation andcomplementation plasmids into a B. burgdorferi B31-A3 strain).Recombination events between the introduced plasmid and the naturallyoccurring plasmid carrying OspA/OspB will result in a certain number ofsuccessful recombinant Borrelia spp. organisms carrying a mutatedOspA/OspB plasmid. Selection can be premised, for example, via a linkedantibiotic resistance and growth in the presence of the correspondingantibiotic. Borrelia spp. organisms with selective elimination ofOspA/OspB expression are contemplated to upregulate the expression ofOspC.

In a further optional embodiment, one or more additional B. burgdorferiss or Borrelia spp. organisms that express OspA, OspB, and/or one ormore additional Osp antigens can be included in the vaccine compositionas additional organisms.

Preferably, each respective inactivated organism is present in thevaccine in a concentration ranging from about 1×10⁴ to about 1×10¹⁰organisms/mL. In particular, the vaccine is preferably administered to acanine in a dose of no less than 1.0×10⁸ organisms/mL of B. burgdorferiss S-1-10, and no less than 5.0×10⁸ organisms/mL of B. burgdorferi ss50772.

Additional OspA Strains

A second strain, providing the OspA antigen, can be a conventionalpathogenic laboratory B. burgdorferi ss isolate (Barbour et al., 1985,J. Clin. Microbiol. 52:478-484) such as B. burgdorferi ss B-31 (ATCC No.35210). A particular second organism is the exemplified B. burgdorferiss S-1-10 strain (ATCC No. PTA-1680). Additional strains suitable foruse as the second organism for vaccine compositions optimized forregions outside of North America include, e.g., the strains: B.burgdorferi ss B-31 (ATCC No. 35210), B. afzelli (e.g., available asATCC No. 51567) and B. garinii (e.g., available as ATCC Nos. 51383 and51991), as well as those listed in Table 1 below.

TABLE 1 Strain Country Cultured from B. burgdorferi ss DK7 ¹ Denmarkskin B. burgdorferi ss 61BV3 ¹ Germany skin B. burgdorferi ss ZS7 ¹Switzerland tick B. burgdorferi ss Pka ¹ Germany tick B. burgdorferi ssIP1, IP2, IP3 ¹ France CSF B. burgdorferi ss HII ¹ Italy blood B.burgdorferi ss P1F ¹ Switzerland synovia B. burgdorferi ss Mil ¹Slovakia tick B. burgdorferi ss 20006 ¹ France tick B. burgdorferi ss212 ¹ France tick B. burgdorferi ss ESP1 ¹ Spain tick B. burgdorferi ssNe-56 ¹ Switzerland tick B. burgdorferi ss Z136 ¹ Germany tick B.burgdorferi ss ia ² Finland CSF ¹ Lagal et al., J. Clin. Microbiol.2003, 41: 5059-5065. ² Heikkila et al, J. Clin. Microbiol. 2002, 40:1174-1180.

The vaccine composition can include a pharmaceutically acceptableadjuvant. “Adjuvants” are agents that nonspecifically increase an immuneresponse to a particular antigen, thus reducing the quantity of antigennecessary in any given vaccine, and/or the frequency of injectionnecessary in order to generate an adequate immune response to theantigen of interest. Suitable adjuvants for the vaccination of animalsinclude, but are not limited to, Adjuvant 65 (containing peanut oil,mannide monooleate and aluminum monostearate); Freund's complete orincomplete adjuvant; mineral gels, such as aluminum hydroxide, aluminumphosphate, and alum; surfactants, such as hexadecylamine,octadecylamine, lysolecithin, dimethyldioctadecylammonium bromide,N,N-dioctadecyl-N′,N′-bis (2-hydroxymethyl)propanediamine,methoxyhexadecylglycerol, and pluronic polyols; polyanions, such aspyran, dextran sulfate, poly IC, polyacrylic acid, and CARBOPOL® (e.g.CARBOPOL 941); peptides, such as muramyl dipeptide, dimethylglycine andtuftsin; and oil emulsions. Information concerning adjuvants isdisclosed, e.g., in the series by P. Tijssen [Practice and Theory ofEnzyme Immunoassays, 3rd Edition, 1987, Elsevier, N.Y., incorporated byreference herein].

Optionally, the vaccine composition can further include apharmaceutically acceptable immune stimulant, such as bacterial and/orfungal cell wall components (e.g., lipopolysaccarides, lipoproteins,glycoproteins, muramylpeptides), various complex carbohydrates derivedfrom plants (e.g., glycans, acemannan), various proteins and peptidesderived from animals (e.g., hormones, cytokines, co-stimulatoryfactors), and novel nucleic acids derived from viruses and/or othersources (e.g., double stranded RNA, CpG).

The vaccine composition is readily administered by any standard routeincluding intravenous, intramuscular, subcutaneous, oral, intranasal,intradermal, and/or intraperitoneal vaccination. The artisan willappreciate that the vaccine composition is preferably formulatedappropriately for each type of recipient animal and mute ofadministration.

Thus, the present invention also provides methods of immunizing a canineagainst B. burgdorferi ss and other Borrelia spp. One such methodcomprises injecting a canine with an immunologically effective amount ofa vaccine of the present invention, so that the canine producesappropriate OspA and OspC borreliacidal antibodies.

In one embodiment, the subcutaneous administration of the presentinvention results in the production of high concentrations of OspA andOspC borreliacidal antibodies including borreliacidal antibodiesspecific for OspC7. In another embodiment, the present inventionprovides a vaccine that comprises a specific, minimum amount of each B.burgdorferi ss strain that is effective against harmful Borrelia spp. Inanother embodiment, a vaccine of the present invention is effective forthe prevention of B. burgdorferi ss and other pathogenic Borrelia spp.infections in dogs. In a particular embodiment, the vaccine comprises asafe and immunologically effective combination of two B. burgdorferi ssstrains of the present invention and a pharmaceutically acceptableadjuvant.

The present invention discloses that a vaccine comprising a specificminimal amount of two B. burgdorferi ss isolates protects dogs fromBorrelia spp. following tick challenge. The present invention furtherdiscloses a vaccine that elicits specific minimal amounts of B.burgdorferi ss-specific OspA and Borrelia spp.-specific OspCborreliacidal antibodies in vaccinated dogs. The vaccines of the presentinvention also may be administered with an acceptable immune stimulantand/or adjuvant.

EXAMPLES

The following examples serve to provide further appreciation of theinvention but are not meant in any way to restrict the effective scopeof the invention.

Example 1 Vaccination with Recombinant OspA

A. Materials and Methods

Animals:

Five- to 10-week old LVG hamsters were obtained from Charles RiverBreeding Laboratories, Inc. (Wilmington, Mass.). Hamsters were housedthree or four per cage at an ambient temperature of 21° C. and providedwith food and water ad libitum.

Vaccination of Hamsters and Collection of Serum:

Hamsters were vaccinated subcutaneously in the back of the neck with 0.5mL of a commercially available recombinant OspA (rOspA) vaccine,RECOMBITEK® Lyme, (Merial Limited, Duluth, Ga.), and given a boosterthree weeks after the primary vaccination. At 3, 7, 9, and 15 weeksafter the primary vaccination, groups of five hamsters each were mildlyanesthetized by inhalation of ether contained in a nose-and-mouth cupand bled by intracardiac puncture. The blood was allowed to clot, andthe serum was separated and stored at −70° C. until use. In addition,hamster sera from three unvaccinated hamsters were pooled and used as anormal serum control. As an additional control, 20 hamsters werevaccinated in both hind thighs with 0.25 mL of a commercially availablewhole cell canine Lyme disease vaccine (Galaxy, Solvay Animal Health,Inc., Mendota Heights, Minn., now Schering-Plough Animal Health,Elkhorn, Nebr.) that contained B. burgdorferi ss S-1-10. The animalswere given a booster, and sera were collected as described above.

Preparation of BSK Medium:

Barbour-Stoenner-Kelly (BSK) broth medium was used for in vitrocultivation of B. burgdorferi and was the primary substrate used in theborreliacidal antibody test and in methods described throughout thepresent application. BSK medium was prepared as described by Callisteret al., [Detection of Borreliacidal Antibodies by Flow Cytometry,Section 11.5.1-11.5.12, Current Protocols in Cytometry, John Wiley andSons, Inc., Supplement 26, (2003) hereby incorporated by referenceherein in its entirety. In brief, the BSK medium was prepared by thefollowing method.

Materials:

HEPES (Sigma)

Neopeptone (Difco)

Sodium citrate (Sigma)

Glucose (Sigma)

Sodium bicarbonate (Sigma)

TC yeastolate (Difco)

Pyruvic acid (Sigma)

N-acetyl glucosamine (Sigma)

Bovine serum albumin (Sigma)

Gelatin (microbiological grade; Difco)

5N NaOH

10× Connaught Medical Research Laboratories (CMRL) 1066

(MP Biomedicals) or Roswell Park Memorial Institute (RPMI) 1640 medium

(Sigma) with L-glutamine and without sodium bicarbonate

Rabbit serum (Life Technologies), heat-inactivated 45 min at 56° C.

56° C. water bath

Positive-pressure pump

Millipore filter manifold

Prefilter (124 mm)

0.2-, 0.45-, and 0.8-μm filters (142-mm diameter)

0.2-μm bell filters

Sterile 100-ml containers

Dark-field microscope

Preparation of BSK Medium

-   1. The following were combined in a 2-liter flask, with from 2 to 4    hours of mixing. 900 mL Mini-Q double-filtered or deionized    distilled water:

6.0 g HEPES

5.0 g neopeptone

0.7 g sodium citrate

5.0 g glucose

2.2 g sodium bicarbonate

2.5 g TC yeastolate

0.8 g pyruvic acid

0.4 g N-acetyl glucosamine

50 g bovine serum albumin (fraction V)

-   2. The components were slowly mixed using the slowest stir plate    setting because vigorous stirring may result in breakdown products    toxic to B. burgdorferi.-   3. While the BSK components were mixing, the gelatin solution was    prepared as follows: in a 500-mL flask, 200 mL Milli-Q    double-filtered or deionized distilled water and 14 g gelatin were    combined and heated on a medium setting with stirring to dissolve.    The solution was then autoclaved for 15 min at 121° C., and then    placed in a 56° C. water bath.-   4. When BSK components were dissolved completely, the solution was    adjusted to pH 7.5 with 5N NaOH.-   5. The 200 mL gelatin solution, 100 mL 10×CMRL 1066 medium, and 64    mL heat-inactivated rabbit serum (45 min at 5600) were then combined    with the other BSK components and thoroughly mixed.    BSK Medium Sterilized-   6. Using the positive-pressure pump, BSK medium was pumped through    the Millipore filter manifold loaded with a 124-mm prefilter and    142-mm 0.2-, 0.45-, and 0.8-μm pore diameter filters stacked from    smallest (bottom) to largest (top) pore size. [The BSK medium cannot    be sterilized by autoclaving. Pre-filtration was necessary to remove    large particulates prior to filter sterilization.]-   7. The BSK medium was then filter-sterilized into a sterile    container using the positive-pressure pump and a sterile 0.2-μm bell    filter.    Sterility Confirmed-   8. A 1-mL aliquot of sterile BSK was aseptically removed and    transferred to a sterile 1.5-mL microcentrifuge tube, and incubated    overnight at 35° C. The remaining filter-sterilized BSK was stored    at 4° C.-   9. Following the incubation, the sterile BSK was examined using    dark-field microscopy to confirm sterility.-   10. The sterile BSK was then transferred into sterile storage    containers. [Headspace was kept to a minimum when filling the    storage containers in order to reduce the oxidation of the sterile    BSK medium.]    Quality Control of Barbour-Stoenner-Kelly (BSK) Medium

Before using BSK medium, it is important to make sure that BSK supportsthe growth of small numbers of Borrelia spp. and that the viablespirochetes grow without clumping. A highly variable component of BSKmedium is bovine serum albumin (BSA). The quality control protocolemployed herein utilized test cultures to incubate and inspect resultingcultures, as described in detail by Callister et al., 2003, Id.

Detection of OspA Borreliacidal Antibodies:

OspA borreliacidal antibodies were detected using a flow cytometricprocedure and B. burgdorferi ss S-1-10 (Callister et al., Arch. Intern.Med. 1994, 154:1625-1632). A fresh culture of the spirochetes wasdiluted with fresh BSK to a concentration of approximately 5×10⁶spirochetes/mL. Concomitantly, serum samples were diluted 1:40 withfresh BSK and sterilized by passage through a 0.2 micron (μm) pore-sizemicrocentrifuge filter. A 200 μL aliquot was then transferred to asterile 1.5 mL screw-cap microcentrifuge tube and serially diluted inBSK from 1:80 to 1:20,480. Serum samples were heat-inactivated at 56° C.for 10 min, and a 100 μL aliquot of the spirochete suspension (5×10⁵spirochetes) and 10 μL of sterile guinea pig complement (Sigma) wereadded. The assays were mixed thoroughly and incubated for 16-24 hours at35° C.

Following incubation, 100 μL of each assay suspension was transferred toa polypropylene tube containing 400 μL of PBS and 1 μg/mL acridineorange. A FACScan flow cytometer (Becton Dickinson ImmunocytometrySystems, San Jose, Calif.) was then used to detect borreliacidalactivity. Spirochetes were isolated by gating (CellQuest software,Becton Dickinson) and analyzed for 1-2 min with the flow rate set atlow. The OspA borreliacidal antibodies were detected indirectly bymonitoring the increased fluorescence intensity that occurs when theacridine orange intercalates into blebbed, non-viable spirochetes. A≧13% shift in the mean fluorescence intensity compared to a normal serumcontrol was considered positive (Callister et al., Clin. Diagn. Lab.Immunol. 2002, 9:908-912). The presence of blebbed, non-motile B.burgdorferi was then confirmed by darkfield microscopy. A positivecontrol was included with each assay, and identical reactivity (+/−onedilution) from run to run was required to minimize interassayvariability. In addition, serum samples collected from each dog wereassayed concurrently.

B. Results

Vaccination with the rOspA vaccine induced only minimal amounts (meantiter <61) of OspA borreliacidal antibodies after 7 weeks that were notdetected after 15 weeks (Table 2). In contrast, hamsters vaccinated withthe canine vaccine that contained a typical (Barbour et al., J. Infect.Dis. 1985, 152-478-484) B. burgdorferi ss strain (S-1-10) induced highlevels of borreliacidal OspA antibodies that peaked at week 7(titer >2560) and remained elevated for the duration of the study.

TABLE 2 Mean titer^(a) of OspA borreliacidal antibodies^(b) aftervaccination with rOspA or GALAXY ™ (S-1-10) canine Lyme diseasevaccines. Week Week Week Week Vaccine 3 7 9 15 rOspA 9 61 4 ND^(c)S-1-10 1470 >2560 970 735 ^(a)Reciprocal dilution. ^(b)Detected by usingB. burgdorferi ss S-1-10. ^(c)ND = None detected.Therefore, the canine recombinant OspA vaccine fails to elicitsignificant titers of OspA borreliacidal antibodies in hamsters.

Example 2 Vaccination with Recombinant OspC

A. Materials and Methods

Animals:

Five- to 10-week old LVG hamsters were obtained from Charles RiverBreeding Laboratories, Inc. (Wilmington, Mass.). Hamsters were housedthree or four per cage at an ambient temperature of 21° C. and providedwith food and water ad libitum.

Preparation of Recombinant (“r”) OspC Vaccine

rOspC was recovered from E. coli JM109 containing pX3-22 as describedpreviously (Rousselle et al., J. Infect. Dis. 1998, 178:733-741).Briefly, the E. coli was cultured at 37° C. in 2×TY broth containingampicillin, and isopropyl-β-d-galactopyranoside (IPTG) (0.1 mM) wasadded during the exponential growth phase. Cells were pelleted bycentrifugation, resuspended in phosphate buffered saline (PBS), andlysed by sonication. Triton X-100 (1% vol/vol) was added, and the lysatewas centrifuged at 10,000×g for 5 m. The sonicated E. coli cells werethen pelleted by centrifugation, and the supernatant was passed over acolumn containing SoftLink resin (Promega) that bound the OspC via abiotinylated purification tag on the amino terminus. The bound OspC wasthen eluted with a purification buffer that also contained 5 mM biotin(Sigma).

Vaccination of Hamsters and Collection of Serum:

Hamsters were vaccinated subcutaneously in the back of the neck with 0.1mL of Freund's complete adjuvant that contained 75 μg of rOspC and thengiven a booster of 75 μg of rOspC in 0.1 mL of Freund's incompleteadjuvant three weeks after the primary vaccination. At 5 weeks after theprimary vaccination, the hamsters were mildly anesthetized by inhalationof ether contained in a nose-and-mouth cup and bled by intracardiacpuncture. The blood was allowed to clot, and the serum was separated andstored at −70° C. until use. In addition, hamster sera from three normalhamsters were pooled and used as a normal serum control.

Detection of OspC Antibodies:

rOspC was diluted to 1000 ng/mL in coating buffer (0.015 M Na₂CO₃, 0.035M NaHCO₃, pH 9.6) and 100 μL amounts were added to individualfat-bottomed amine-binding microtiter wells (Costar, Cambridge, Mass.).Microtiter plates were incubated overnight at 4° C. After incubation,plates were washed 3 times with PBS (pH 7.2) and blocked with PBScontaining 0.05% TWEEN 20 (Sigma) and 1% bovine serum albumin (Sigma)for 1 hour at room temperature with shaking. After blocking, plates werewashed again with PBS. Subsequently, 100 μL amounts of hamster serumserially diluted from 1:80 to 1:20,480 in PBS/Tween was added toindividual wells, and the plates were incubated for 1 hour at roomtemperature. Following incubation, plates were washed three times withPBS, 100 μL of anti-hamster IgG horseradish peroxidase conjugate(Organon Teknika Cappel) diluted 1:3000 in PBS/Tween was added to eachwell, and the plates were re-incubated at room temperature for 1 hour.Plates were then washed 3 times with PBS and 100 μL ofo-phenylenediamine phosphate (0.4 mg/ml Sigma) was added to each welland allowed to incubate at room temperature for 30 min, Reactions werestopped by adding 100 μL of 1 N H₂SO₄ and absorbances at 490 nm (modelEL 311; Bio-Tek Inc, Winooski, Vt.) were immediately determined.

Detection of OspC Borreliacidal Antibodies:

OspC borreliacidal antibodies were detected as described above (Example1), except B. burgdorferi ss 50772 was used.

B. Results

Vaccination with the rOspC vaccine induced high levels of OspCantibodies detected by the ELISA (Table 3, infra), but only a low level(titer 80) of borreliacidal activity was present. The vaccination withrOspC had therefore induced high concentrations of OspC antibodies, butthe response was comprised almost entirely of antibodies (eg.opsonizing) that would not provide protection against infection. Thiswas especially significant when one considered the 75 μg concentrationof proteins in the B. burgdorferi ss 50772 vaccine was calculatedwithout accounting for the presence of numerous additional non-OspCproteins. The collective results (Examples 1 and 2) therefore confirmedprevious findings (Straubinger et al. Vaccine 2003, 20:181-193) thatrecombinant Osps stimulated significantly less amounts of borreliacidalantibodies than intact B. burgdorferi ss.

TABLE 3 Mean titer^(a) of antibodies detected by OspC ELISA or OspCborreliacidal antibody test^(b) after vaccination with rOspC. Antibodytiter detected by: Sample OspC ELISA OspC borreliacidal antibody testNormal ND^(c) ND rOspC 10240 80 ^(a)Reciprocal dilution of pooled serumfrom 5 hamsters. ^(b)Detected by using B. burgdorferi ss 50772 ^(c)ND =None detected.Therefore, the canine recombinant OspC vaccine fails to elicitsignificant titers of OspC borreliacidal antibodies in hamsters.

Example 3 Vaccination with B. burgdorferi ss Isolate 5-1-10

A. Materials and Methods

Organism:

B. burgdorferi ss S-1-10 is a typical pathogenic strain generallyindistinguishable from B. burgdorferi ss B31 (Barbour et al., J. Infect.Dis., 1985, 152:478-484). The isolate was originally recovered by Dr.Steven M. Callister, Gundersen Lutheran Medical Foundation, La Crosse,Wis., from the kidney of a white-footed mouse, Peromyscus leucopus,captured February 1988 from a site near La Crosse, Wis., and waslicensed by Solvay Animal Health for incorporation into a canine Lymedisease vaccine. The subsequent commercial product (GALAXY™ Lyme) wasacquired by Schering Plough Animal Health on Apr. 17, 1997. The organismexpresses OspA, OspB, and OspC. However, as is typical of infectious B.burgdorferi ss isolates, significantly higher concentrations of OspA andOspB are produced when the spirochetes are cultured in laboratory BSKmedium. It has been shown that the expression of OspC can be maximizedby incubation at 35° C. (Schwan, Biochem. Soc. Trans. 2003, 31:108-112).

Animals

Eight-week-old beagle puppies (Ridglan Farms, Mount Horeb, Wis.) werehoused communally, and food and water was available ad libitum. Theexperiment was reviewed and approved by the Schering Plough AnimalHealth Animal Care and Use Committee (IACUC).

Preparation of B. burgdorferi ss S-1-10 Vaccine:

A fresh culture of B. burgdorferi ss S-1-10 that had reached logarithmicgrowth phase by incubation in BSK at 35° C. was inactivated by addingbinary ethylenimine (BEI) to a final concentration of 10 mM andincubating for an additional 48 hours. After inactivation, the BEI wasneutralized by adding sterile sodium thiosulfate and incubating at 35°C. for 6 to 12 hours. The spirochetes were then pelleted bycentrifugation and resuspended in sterile balanced salt solutioncontaining ≦30 μg of gentamicin/mL and ≦30 units of nystatin/mL. Thespirochetes were then blended with 5% Emulsigen® (MVP Laboratories,Inc.) and 1% HEPES so that a 1.0 mL dose contained ≧2.5×10⁷ spirochetes.

Vaccination and Collection of Serum:

Dogs were vaccinated subcutaneously in the neck with a 1 mL dose of theS-1-10 vaccine and boosted with an additional 1 mL dose after 21 days.Whole blood was collected immediately prior to the booster vaccination(study day 21) and at study days 28, 35, 47, 83, and 113 by venipunctureof the jugular vein. The serum was separated by centrifugation andstored at −20° C. until tested.

Detection of Borreliacidal Antibodies:

OspA and OspC borreliacidal antibodies were detected by flow cytometryemploying B. burgdorferi ss S-1-10 and 50772, as described above inExamples 1 and 2.

B. Results

Vaccination with B. burgdorferi ss S-1-10 reliably induced high levelsof borreliacidal OspA antibodies that were detectable at study day 21,peaked at study day 28, and remained detectable at study day 113 (Table4, infra). In contrast, OspC borreliacidal antibodies were not detected(titer less than 1:80). Thus, the conventional B. burgdorferi ss strainfailed to induce the production of OspC borreliacidal antibodies despitemaximizing the OspC expression by incubating the spirochetes at 35° C.

TABLE 4 Mean titers^(a) (n = 8) of OspA or OspC borreliacidal antibodiesafter vaccination with B. burgdorferi ss S-1-10. Borreliacidal Day DayDay Day Day Day Antibody 21 28 35 47 83 113 OspA^(b) 202 9481 9481 47401613 1382 OspC^(c) ND^(d) ND ND ND ND ND ^(a)Reciprocal dilution.^(b)Detected by using B. burgdorferi ss S-1-10. ^(c)Detected by using B.burgdorferi ss 50772. ^(d)ND = None detected.Therefore, a vaccine employing B. burgdorferi ss isolate S-1-10 failedto elicit a significant titer of OspC borreliacidal antibodies.

Example 4 Vaccination with B. Burgdorferi ss Isolate 50772

A. Materials and Methods

Organism:

B. burgdorferi ss 50772 is a unique ospA-ospB-strain originally reportedby Anderson et al., J. of Clan. Microbiol., 1996, 34:524-529, who failedto identify it as expressing OspC antigen. The 50772 strain is describedby U.S. Pat. No. 6,464,985, incorporated herein by reference in itsentirety, as expressing OspC antigen, and this strain is reported bythat U.S. patent as deposited with the ATCC as No. PTA-439.

Animals:

Eight-week-old beagle puppies (Ridglan Farms, Mount Horeb, Wis.) werehoused communally, and food and water was available ad libitum. Theexperiment was reviewed and approved by the Schering Plough AnimalHealth Animal Care and Use Committee (IACUC).

Preparation of B. burgdorferi ss 50772 Vaccine:

B. burgdorferi ss 50772 vaccine was prepared as described above inExample 3.

Vaccination and Collection of Serum:

Dogs were vaccinated subcutaneously in the neck with a 1 mL dose of the50772 vaccine and boosted with an additional 1 mL dose after 21 days.Whole blood was collected immediately prior to the booster vaccination(study day 21) and at study days 28, 35, 47, 83, and 113 by venipunctureof the jugular vein. The serum was separated by centrifugation andstored at −20° C. until tested.

Detection of Borreliacidal Antibodies:

OspC borreliacidal antibodies were detected by flow cytometry employingB. burgdorferi ss 50772 as described above in Examples 1 and 2.

B. Results

Vaccination with B. burgdorferi ss 50772 reliably induced high levels ofborreliacidal OspC antibodies that peaked at study day 35 and remaineddetectable at study day 113 (Table 5, infra). Thus, in contrast tovaccinations with rOspC or a traditional B. burgdorferi ss isolate(S-1-10), vaccination with the unique B. burgdorferi ss strain 50772induced significant levels of OspC borreliacidal antibodies.

TABLE 5 Mean Titers^(a) (n = 8) of OspC Borreliacidal Antibodies^(b)After Vaccination with B. burgdorferi ss 50772. Day 21 Day 28 Day 35 Day47 Day 83 Day 113 ND^(c) 5530 1097 435 274 202 ^(a)Reciprocal dilution.^(b)Detected by using B. burgdorferi ss 50772. ^(c)ND = None detected.Therefore, a vaccine comprising the B. burgdorferi ss isolate 50772induces high concentrations of OspC borreliacidal antibodies.

Example 5 Preparation of a Vaccine Comprising B. burgdorferi ss IsolatesS-1-10 and 50772

A. Materials and Methods

Growth:

Frozen stock cultures of B. burgdorferi ss S-1-10 and B. burgdorferi ss50772 were cultured under standard conditions, i.e., the stock cultureswere grown at 33±2° C. and 35±2° C., respectively, in individual screwcap culture tubes containing 5 to 17 mL of BSK (Barbour Stoenner Kelly)medium [prepared according to Callister et al., 1990, J. of ClinicalMicrobiology 28: 363-365, hereby incorporated by reference in itsentirety herein] under an atmosphere of air enriched with 5% CO₂. Afterreaching logarithmic growth, the cultures were used to inoculate 150 to200 mL of fresh BSK medium, which were next incubated until the culturesreached logarithmic growth. The resultant suspensions were then used toinoculate 5 to 10 liters of fresh BSK contained in 10 to 20 liter jugs(production culture) prior to an additional incubation, as describedabove.

Inactivation:

The spirochetes in the production cultures were inactivated by addingBEI to a concentration of 10 mM and incubating with slow stirring for 24to 48 hours. After inactivating the spirochetes, the BEI was neutralizedby adding 10.6 mL of sterile 3.015M sodium thiosulfate to each liter ofBSK/spirochete suspension and incubating with slow stirring for 6 to 12hours.

Concentration and Blending:

Inactivated spirochetes were pelleted by centrifugation and resuspendedin a sterile balanced salt solution containing 30.0 μg/mL of gentamicinand 30 units/mL of nystatin. The test vaccine was blended with 5%Emulsigen® (MVP Laboratories, Inc.) and filled in 2.0 mL glass vials soeach dose (1.0 mL) contained ≧2.5×10⁷ organisms/mL of B. burgdorferi ssS-1-10, ≧5.0×10⁸ organisms/mL of B. burgdorferi ss 50772, 1% HEPES, 29μg/mL gentamicin, and 29 units/mL nystatin.

Example 6 Safety and Efficacy of a Vaccine Comprising B. burgdorferi ssIsolates S-1-10 and 50772

A. Materials and Methods

Animals:

Eight-week-old beagle puppies (Ridglan Farms, Mount Horeb, Wis.) werehoused individually or communally, and food and water was available adlibitum. The experiment was reviewed and approved by the Schering PloughAnimal Health Animal Care and Use Committee (IACUC).

Vaccination and Collection of Sera:

Dogs were vaccinated subcutaneously in the neck with a 1 mL dose of thevaccine and boosted with an additional 1 mL dose after 21 days. Wholeblood was collected prior to the initial (study day −3) and boostervaccination (study day 21) and also on study days 28, 35, 43, 78, 106,134, 162, and 197 by venipuncture of the jugular vein. The serum wasseparated by centrifugation and stored at −20° C. until tested.

Post-Vaccination Observations:

After each vaccination, injection sites were palpated daily until noreaction could be felt, and rectal temperatures were recorded 4-6 hourspost-vaccination and on days 1-3 post-vaccination.

Tick Challenge:

Three weeks after the second vaccination, dogs were shaved on the rightside of the thoracic cavity, and 10 female and 10 male B. burgdorferiss-infected I. scapularis ticks were placed in a rubber cup that wassecured to the shaved area with tape and bandage wrap. The ticks wereallowed to feed for 7 days.

Detection of B. Burgdorferi ss in Ticks:

The tick mouthparts were removed by scalpel, and the midguts were teasedand smeared onto glass slides and allowed to dry overnight at roomtemperature. After drying, the slides were fixed in acetone for 8-10 minand air-dried. Species-specific mouse OspA monoclonal antibody H5332 wasdiluted 1:40 in PBS (pH 7.2) and overlaid with goat anti-mousefluorescein isothiocyanate-labeled immunoglobulin G antibody diluted1-500 in PBS. After incubation, slides were rinsed with PBS, air-dried,and examined by fluorescence microscopy. Each slide was examinedindependently by two experienced microbiologists.

Blood Samples:

Whole blood was collected in serum separation transport (SST) tubes onstudy days 78, 106, 134, 162, and 197, and the serum was separated andstored at −20° C. until tested.

Removal of OspA Antibodies:

Recombinant (r)OspA was recovered from Escherichia coli (“E. coli”) DH5αexpressing OspA-glutathione-S-transferase fusion protein as describedpreviously (Callister et al., J. Infect. Dis. 1993, 167:158-164).Briefly, the E. coli was cultured at 37° C. in 2×TY broth containingampicillin, and isopropyl-β-d-galactopyranoside (“IPTG”) (0.1 mM) wasadded during the exponential growth phase. Cells were pelleted bycentrifugation, resuspended in phosphate buffered saline (“PBS”), andlysed by sonication. Triton X-100 (1% vol/vol) was added, and the lysatewas centrifuged at 10,000×g for 5 minutes (min.). The supernatantcontaining the fusion protein was then passed over aglutathione-Sepharose 4B column (Pharmacia), and the recombinant OspAwas eluted with 50 mM Tris-Cl (pH 8.0) plus 5 mM reduced glutathione andresuspended in purification buffer (50 mM Tris [pH 8], 50 mM NaCl, 2 mMEDTA, 0.1% Triton X-100).

The rOspA fusion protein was then bound to Sepharose 4B by cyanogenbromide (CNBr) activation. Specifically, 0.8 g of CNBr-activatedSepharose 4B (Pharmacia) was washed with coupling buffer (0.1 MNaHCO₃-0.5 M NaCl, pH 8.3), a 2 mg amount of OspA in coupling buffer wasadded to the gel, and the mixture was gently shaken at room temperaturefor 2 hours. After the gel was washed twice with coupling buffer, 4 mLof ethanolamine (pH 9) was added, and the gel was incubated for 2 hoursto block unbound sites. The gel was washed three times with 50 mL of 0.1M sodium acetate-0.5 M NaCl (pH 4.0) and then equilibrated in PBS. A 1mL sample of immune serum diluted ten-fold in PBS was passed over thecolumn four times.

Removal of OspC Antibodies:

rOspC was recovered from E. coli JM109 containing pX3-22 as describedpreviously (Rousselle et al., J. Infect. Dis. 1998, 178:733-741).Production of the recombinant protein was induced as described for rOspAabove. The sonicated E. coli cells were then pelleted by centrifugation,and the supernatant was passed over a column containing SoftLink resin(Promega) that bound the OspC via a biotinylated purification tag on theamino-terminus. The bound OspC was then eluted with a purificationbuffer that also contained 5 mM biotin (Sigma).

A 1 mL volume of Tetralink tetrameric avidin resin (Promega) was thenwashed, suspended in 40 mL of PBS, and loaded onto a 10-mm by 70-mmpolypropylene column. A 0.5 mg amount of dialized rOspC in a 1 mL volumeof PBS was passed over the column and binding was confirmed by proteinassay (Bio-Rad). A 1 mL volume of immune serum diluted ten-fold in PBSwas passed over the column four times.

Removal of OspC7-Specific Antibodies:

A fusion protein containing the 7 C-terminal amino acids of OspC (OspC7)was recovered from E. coli JM109 containing pXT7 as described previously(Lovrich et al., Clin. Diagn. Lab. Immunol. 2005, 12:746-751,incorporated by reference herein in its entirety). Production of therecombinant protein was induced as described for rOspA above. Thesonicated E. coli cells were then pelleted by centrifugation, and thesupernatant was passed over a column containing SoftLink resin (Promega)that bound the OspC7 via a biotinylated purification tag on theamino-terminus. The bound OspC7 was then eluted with a purificationbuffer that also contained 5 mM biotin (Sigma). A 1 mL volume ofTetralink tetrameric avidin resin (Promega) was then washed, suspendedin 40 mL of PBS, and loaded onto a 10-mm by 70-mm polypropylene column.A 0.5 mg amount of dialyzed rOspC in a 1 mL volume of PBS was passedover the column and binding was confirmed by protein assay (Bio-Rad), A1 mL volume of immune serum diluted tenfold in PBS was passed over thecolumn four times.

Detection of Borreliacidal Antibodies:

OspA and OspC borreliacidal antibodies were detected by flow cytometryemploying B. burgdorferi ss S-1-10 and 50772 as described above inExamples 1 and 2.

Skin Biopsies:

Skin biopsies were taken from sites adjacent to tick attachment on studydays 78, 106, 134, 162, and 197 with a disposable 4 mm puncture device.The biopsies were placed into separate tubes containing 9 mL of BSK(Callister et. al, J. Clin. Microbiol. 1990, 28:363-365) supplementedwith gelatin (BSK+G), 40-50 micrograms (μg)/mL rifampin, and 8 μg/mLkanamycin. Cultures were incubated at 35±2° C. for 3 weeks and examinedweekly by dark-field microscopy for spirochetes.

Immune Suppression:

Dogs were immune suppressed by daily administration of dexamethasone(0.4 mg/lb body weight) for 5 days beginning 13 weeks post-challenge.

Clinical Observations:

Dogs were observed daily for limb/joint disorders including stiffness(reluctant to put full weight on limbs), limping (favoring a limb whilewalking or running), or lameness (non-weight bearing). Scoring was madeby the consensus of at least two observers, and dogs were examined bythe Attending Veterinarian to rule out any other external injuries.

Necropsy:

Dogs that had limb/joint disorders for at least 3 consecutiveobservation periods were euthanized and necropsied. Tissue samples fromthe elbow, carpus, knee, tarsus, heart, spleen, bladder, and bothkidneys were collected and processed for isolation of B. burgdorferi byculture. Joint tissues were cultured in individual tubes containing 9 mLBSK+G. Other tissues (heart, spleen, urinary bladder, and kidneys) werecombined with 9 mL BSK+G, homogenized thoroughly with a stomacher, and a1 mL amount was transferred to a separate tube containing 9 mL of freshBSK+G. Cultures were incubated at 35±2° C.

The remaining dogs were euthanized and necropsied at the end of thestudy. Tissue samples from these dogs were collected from the jointcapsule and tendon of the elbow, carpus, knee, and tarsus and stored in10% buffered formalin for histopathology examination. In addition,samples from the joint capsules of the elbow, carpus, knee, and tarsuswere placed into individual tubes containing 9 mL BSK+G or BSK+G withantibiotics and incubated at 35±2° C. for 3 weeks.

Western Blotting:

Western blotting was performed using standard techniques. B. burgdorferiss 297 was boiled in sample buffer for 5 min and 150 μg of total proteinwas loaded onto a 0.1% SDS-12% polyacrylamide gel (4% polyacrylamidestacking gel without comb). Protein concentrations were determined witha protein determination kit, and two gels were run simultaneously in anelectrophoresis unit. After electrophoresis, proteins were transferredto nitrocellulose, and the nitrocellulose was cut into strips andblocked with PBS-0.3% TWEEN 20 for 30 min at 22° C. Strips wereincubated for 1 hour at 22° C. with dog serum diluted 1:100 and washedthree times with PBS-0.05% TWEEN 20. Horseradish peroxidase-labeledanti-dog IgG (Kirkegaard & Perry Laboratories, Gaithersburg, Md.) wasadded, and the strips were incubated for 30 min at 22° C. beforedeveloping with the TMB Membrane Peroxidase Substrate System (Kirkegaard& Perry Laboratories, Gaithersburg, Md.).

B. Results

Safety of the Vaccine:

Dogs remained clinically normal following vaccination, including noincreases in rectal temperature. Dogs vaccinated with the vaccine didhowever develop slight swelling at the injection site that resolvedwithin 8 days. The largest reaction measured 1×1×0.5 cm (24 hourpost-vaccination).

Post-Vaccination Serology:

Vaccination with placebo failed to induce antibodies specific for B.burgdorferi ss. In contrast, vaccination with the vaccine inducedsignificant levels of borreliacidal antibodies detected by using B.burgdorferi ss isolates S-1-10 or 50772. The mean titer of borreliacidalantibodies detected by using S-1-10 peaked one week after the boostervaccination (study day 28) and remained detectable at study day 197(Table 6 below). Similarly, high concentrations of borreliacidalantibodies specific for isolate 50772 were present by study day 28 andremained elevated at study day 43 and detectable at study day 197 (Table7 below).

TABLE 6 Mean titers^(a) (n = 15) of borreliacidal activity^(b) aftervaccination with the vaccine. Day Day Day Day Day Day Day Day Day DayGroup −3 21 28 35 43 78 106 134 162 197 VaccinatesND^(c) >211 >8128 >6160 >4889 1404 970 970 1016 220 Controls ND ND ND NDND ND ND ND ND ND ^(a)Reciprocal dilution. ^(b)Detected by usingborreliacidal antibody test with B. burgdorferi ss S-1-10. ^(c)ND = Nonedetected.

TABLE 7 Mean titers^(a) (n = 15) of borreliacidal activity^(b) aftervaccination with the vaccine. Day Day Day Day Day Day Day Day Day DayGroup −3 21 28 35 43 78 106 134 162 197 Vaccinates ND^(c) ND 1470 884221 50 48 46 42 42 Controls ND ND ND ND ND N/A^(d) N/A N/A N/A N/A^(a)Reciprocal dilution. ^(b)Detected by using borreliacidal antibodytest with B. burgdorferi ss 50772. ^(c)ND = None detected. ^(d)N/A = Notapplicable.

Confirmation of OspA and OspC Borreliacidal Antibodies.

To confirm that the borreliacidal activity was due to OspA and OspCborreliacidal antibodies, Immune sera from five dogs vaccinated with thevaccine were passed over separate columns containing rOspA or rOspC andthe effects on the borreliacidal activities were examined. RemovingOspA-specific or OspC-specific antibodies by adsorbing the sera with therecombinant proteins significantly (≧4-old reduction) decreased theborreliacidal activity detected by using the B. burgdorferi ss S-1-10 or50772 isolates respectively. The collective findings therefore confirmedthe vaccine induced significant levels of OspA and OspC borreliacidalantibodies, and the borreliacidal activities detected by the B.burgdorferi ss S-1-10 or 50772 were almost entirely specific OspA orOspC borreliacidal antibodies, respectively. See Table 8 below.

TABLE 8 Effect of removing OspA or OspC antibodies on borreliacidalactivity in sera from dogs (n = 5) vaccinated with the vaccine.Borreliacidal Antibody Titer^(a): OspA^(b) OspC^(c) before after beforeafter Serum absorption absorption absorption absorption 1 5120 160 5120640 2 2560 160 5120 320 3 10240 ND^(d) 5120 320 4 2560 ND  1280 160 51280  80 2560 320 OspA control 2560 ND — — OspC control — — 2560 ND^(a)Reciprocal dilution. ^(b)Sera collected on study day 43. ^(c)Seracollected on study day 28. ^(d)ND = None detected.

Confirmation of OspC7-Specific Borreliacidal Antibodies

To confirm that the OspC borreliacidal antibodies contained asignificant proportion of borreliacidal antibodies specific for OspC7,immune sera from five dogs vaccinated with the vaccine were passed overa column containing rOspC7, and the effects on the borreliacidalactivity were examined. Removing the OspC7-specific antibodies byadsorbing the sera with the recombinant OspC7 protein significantly (2-to 4-old reduction) decreased the borreliacidal activity detected byusing the B. burgdorferi ss 50772 isolate. Therefore, the findingsconfirmed that the vaccine induced significant levels of OspCborreliacidal antibodies specific for the conserved OspC7 epitope. SeeTable 9 below.

TABLE 9 Effect of removing OspC7 antibodies on borreliacidal activity insera from dogs (n = 5) vaccinated with the vaccine. BorreliacidalAntibody Titer^(a): Serum before absorption after absorption 1 204805120 2 10240 5120 3 10240 2560 4 5120 2560 5 10240 2560 OspC control2560 ND ^(a)Reciprocal dilution. ^(b)Sera collected on study day 28.^(c)ND = None detected.

Ability of the OspA and OspC Borreliacidal Antibodies to SterilizeInfected Ticks:

Examining the midguts of the ticks that fed on the vaccinated or controldogs confirmed that the OspA and OspC borreliacidal antibodies inducedby the vaccine had sterilized the ticks. B. burgdorferi ss were detectedin the tick smears from 34 (32%) of 106 female ticks that had fed on 13of 15 placebo-vaccinated dogs (Table 10 below). In contrast, nospirochetes (0 of 99) were detected in the midguts from the female ticksthat fed on dogs vaccinated with the vaccine (p<0.0001).

TABLE 10 Detection of B. burgdorferi ss in female ticks removed fromvaccinated or control dogs. Female Ticks Processed Smears PositiveTreatment (Engorged and Not for Total No. of Group engorged) B.burgdorferi Dogs Positive Vaccinates  99/151 0/99 0/15 (61%) (0%)Controls 106/148 34/106 13/15* (64%) (32%)  *p < 0.0001Ability of the Vaccine to Prevent Recovery of B. Burgdorferi ss from theSkin:

The vaccine-induced borreliacidal antibodies also prevented thespirochetes from colonizing the skin, B. burgdorferi ss were recoveredfrom 56 (79%) of 71 biopsies collected from the placebo-vaccinated dogsat monthly intervals following tick-challenge (Table 11 below) andspirochetes were recovered from at least one skin biopsy from 14 (93%)of the 15 dogs. In contrast, B. burgdorferi ss were not recovered fromany skin biopsies collected from the dogs vaccinated with the vaccine(p<0.0001).

TABLE 11 Isolation of B. burgdorferi ss from the skin of vaccinated ornon-vaccinated control dogs. Total Total No. Treatment Day Day Day DayDay Biopsies of Dogs Group 78 106 134 162 197 Positive PositiveVaccinated  0/15  0/15  0/15  0/15 0/13  0/73 0/15 (0%) (0%) Controls12/15 13/15 11/15 11/14 9/12 56/71 14/15* (79%)  (93%) P < 0.0001

Ability of the Vaccine to Prevent Serologic Evidence of Infection:

The vaccine also prevented the development of Lyme disease-specificantibodies. A previous investigation (SPAHC Report B01-184-01R)confirmed that infection with B. burgdorferi ss reliably induced dogantibodies that bound an approximately 20 kDa protein, and the responsewas not induced by the vaccine (FIG. 1). In the current study,antibodies that bound the infection-specific 20 kDa protein weredetected readily in the immune sera from 14 (93%) of the 15placebo-vaccinated control dogs (FIG. 2A). In addition, the serum thatdid not contain any 20 kDa protein antibodies was collected from thecontrol dog that also failed to yield spirochetes from the skin. Incontrast, 20 kDa protein antibodies were not produced (p<0.0001) by anyof the dogs vaccinated with the vaccine (FIG. 2B).

In addition, dogs with Lyme disease produce non-OspC borreliacidalantibodies that are also detectable by using the B. burgdorferi ss 50772isolate (Callister et al., J. Clin. Microbiol, 2000, 38:3670-3674). Thespecific target of the response is unknown, but the antibodies providehighly specific serodiagnostic confirmation of infection. Dogsvaccinated with the vaccine were already producing OspC borreliacidalantibodies, but no dogs developed a significant (≧4-fold) increase inthe level of B. burgdorferi ss 50772-specific borreliacidal antibodiesafter the tick challenge. It was therefore highly unlikely thevaccinated dogs were infected with spirochetes. In contrast,borreliacidal antibodies were not produced by the placebo-vaccinateddogs prior to challenge, but 10 (67%; p=0.0002) of the 15 dogs developedsignificant levels (titers ≧1:640) of borreliacidal antibodies detectedby using B. burgdorferi ss 50772 after the ticks were allowed to feed(Table 12 below).

TABLE 12 Mean titers^(a) of borreliacidal antibodies^(b) after tickchallenge. Total No. of Dogs with Treatment Day Day Day Day Day IncreaseGroup 78 106 134 162 197 Titers Vaccinates 50 48 46 42 42 0/15Controls >121 >463 >640 >390 >403 10/15* *p = 0.0002 ^(a)Reciprocaldilution. ^(b)Detected by using B. burgdorferi ss 50772.

Ability of the Vaccine to Prevent Frank Limb/Joint Disorders:

Previous studies (Summers et al., J. Comp. Path. 2005, 133:1-13, Wikleet al., J. Appl. Res. Vet. Med. 2006, 4:23-28) demonstrated thatinfection with B. burgdorferi ss only rarely cause frank limb/jointdisorders (e.g. swelling, lameness) and our findings confirmed this.Only one (8%) placebo-vaccinated control dog developed a jointabnormality. The left front leg was stiff 9 weeks after thetick-challenge, and B. burgdorferi ss spirochetes were recovered fromthe elbow. However, the findings were not significant because two dogsvaccinated with the vaccine also developed stiffness in one or morelimbs, but, in contrast to the dog that received placebo spirocheteswere not recovered from any tissues.

To exacerbate the development of frank symptoms, the remaining dogs wereimmunosuppressed. Subsequently, three placebo-vaccinated control dogsbecame lame, and B. burgdorferi ss were recovered from two of thesedogs. In contrast, no immunosuppressed dogs vaccinated with the vaccinedeveloped lameness. Thus, the collective findings suggested the vaccineprevented the development of Lyme arthritis, although the results werenot significant (p=0.0996). However, see the additional data presentedand discussed below.

Ability of the Vaccine to Prevent Erosive Changes Associated with B.burgdorferi ss Infection:

When the joints of the dogs discussed above were examinedmicroscopically, however, the findings clearly demonstrated theeffectiveness of the vaccine. The joint capsules from the remaining dogs(n=13) vaccinated with the vaccine were normal. In contrast, one or morejoint tissues from 6 (p=0.0034) of the 11 remaining placebo-vaccinatedcontrol dogs (Table 7 above) had significant inflammation characterizedby infiltration of neutrophils and mononuclear cells (FIG. 3). Moreover,B. burgdorferi ss were not recovered from any tissues from the dogsvaccinated with the vaccine, but spirochetes were recovered from jointtissues from 5 (83%) of the 6 placebo-vaccinated control dogs witherosive changes. The collective findings therefore, confirmed thevaccine prevented canine Lyme arthritis and also corroborated previousreports that the syndrome is characterized most often by subclinicalsynovitis (Summers et al., J. Comp. Path. 2005, 133:1-13; Wikle et al.,J. Appl. Res. Vet Med. 2006, 4:23-28).

Example 7 Specificity of Naturally-Occurring Borreliacidal AntibodiesDirected to the OspC7 Epitope

Data confirming that borreliacidal antibodies elicited in a mammalagainst OspC7 are specific have been obtained by analyzing sera fromindividuals (human) with and without a history of Lyme disease orLyme-related symptoms, as follows.

A. Materials and Methods

1. Sera:

Normal sera from individuals with no previous history (chart-review) ofLyme disease or Lyme-related symptoms (n=36), uncharacterized sera fromblood donors (n=100) or individuals undergoing cholesterol screenings(n=100), or sera from volunteers with blood factors or illnesses thatcommonly cross-react with B. burgdorferi ss antigens, includingantinuclear antibodies (n=20), rheumatoid factor (n=20), mononucleosis(n=10), cytomegalovirus (n=10), syphilis (n=13) or Rocky Mountainspotted fever (n=4), were from archived samples stored at −20° C. Lymedisease sera were collected from patients evaluated at GundersenLutheran Medical Center during 2003 or 2004. Sera (n=86) from patientswith erythema migrans (EM) that fulfilled the CDC surveillance criterion(Centers for Disease Control and Prevention. Morb. Mort. Wkly. Rep.1990, 39:19-21.) were classified as likely Lyme, sera (n=22) frompatients with tick exposures and atypical skin lesions were classifiedas probable Lyme (n=49), and sera from patients with tick exposure andconstitutional symptoms that included primarily headache, fever,myalgia, and arthralgia were classified as possible Lyme. The sera werecollected during the initial visit, stored at −20° C., and blinded priorto testing.

OspC7 peptide. The 7-aa OspC7 peptide (AESPKKP; SEQ ID NO: 1) wassynthesized at the University of Wisconsin Biotechnology Center(Madison, Wis.) by using an automated synthesizer (Protein Technologies)and the Fmoc method (Fields, et al., Peptide Res. 1991, 4:95-101).Following synthesis, the amino-terminal end of the peptide wasbiotinylated manually by HBTU activation and purified by high-pressureliquid chromatography. Composition was confirmed using matrix-assistedlaser desorption ionization-time of flight (MALDI-TOF) mass spectrometry(predicted mass 1095.4; observed mass 1095.8).

OspC7 ELISA. Individual wells of microtiter plates (Immunolon 2 HB,Thermo Labsystems, Franklin, Mass.) were coated with 100 μL of a 4 μg/mlsuspension of streptavidin (Pierce, Rockland, Ill.) contained incarbonate buffer (90 mM NaHCO₃, 60 mM Na₂CO₃; pH 9.6) and incubatedovernight at 4° C. Following incubation, plates were washed five timeswith Tris-buffered saline (TBS-T; 13 mM Tris HCl, 3 mM Tris base, 140 mMNaCl, 2.7 mM KCl; pH 7.4) containing 0.05% Tween 20. After washing, 200μL of blocking buffer (15 mM NaCl, 10 mM Tris HCl, 3% fetal bovineserum, 0.05% Tween 20) containing 1 μg/mL of biotinylated OspC7 peptidewas added to each well and incubated with rotation (150 rpm) for 1 hourat room temperature, Plates were washed three times with TBS-T andreacted with 100 μL amounts of sera diluted 1:200 in blocking buffer for1 hour at room temperature. The secondary antibody wasperoxidase-conjugated goat anti-human IgM and IgG (Kirkegaard PerryLaboratories, Gaithersburg, Md.) diluted 1/15,000 in blocking buffer.After incubation for one hour, the bound secondary antibody wasquantified by the addition of o-phenylenediamine and hydrogen peroxidein citrate buffer (Sigma, St. Louis, Mo.) and determination of the OD at490 nm (SpectraMax 250; Molecular Devices, Sunnyvale, Calif.).

B. Results

In previous studies (Jobe et al., Clin. Diagn. Lab. Immuno. 2003,10:573-578, Lovrich et al., Clin. Diagn. Lab. Immunol. 2005,12:746-751), a highly conserved immunodominant OspC borreliacidalantibody epitope was localized to the region within the 7 amino acidsnearest the C-terminus (OspC7). To confirm the epitope inducedantibodies specific to infection with Borrelia spp., the reactivities ofan ELISA that used OspC7 using sera from patients with Lyme disease(FIG. 5) and sera from normal subjects or patients with other illnesseslikely to produce antibodies that could also bind Borrelia spp. proteins(FIG. 4) were compared. In FIGS. 4 and 5, the line at Absorbance 1.25defines three standard deviations above the mean absorbance of thenormal and potentially cross-reactive sera. Any results that fall abovethe line therefore have a 99% probability that the reactivity issignificant (true positive). Significant reactivity was detected onlyrarely in the normal or potentially cross-reactive sera (FIG. 4). Itshould also be noted that the majority of the positive results wereobtained using sera that could have come from Lyme disease patients,because the sera (CS) were obtained from a group of 100 patients beingseen at Gundersen Lutheran Medical Center for a variety of ailments. Theregion is a highly endemic focus of Lyme disease, and the identities andclinical histories of the patients were unknown. In contrast, positivesera were not detected in serum samples (n=100) collected randomly fromblood donors (BD) in Milwaukee, a non-endemic region of Wisconsin.

In addition, positive results (greater than 3 standard deviations abovethe mean of the sera that should be non-reactive—FIG. 4) were detectedcommonly in the sera from the Lyme disease patients and the absorbancevalues were high (FIG. 5). The collective findings therefore confirmedthe high specificity of the antibodies directed against the OspC7epitope and the immunodominance of the response during human Lymedisease.

Summarizing the above-exemplified data, groups (15 dogs) of 8 week-oldpuppies were vaccinated and boosted with a vaccine that contained5.0×10⁸ B. burgdorferi ss 50772, 2.5×10⁷ B. burgdorferi S-1-10 ss and 5%Emulsigen® or a placebo that contained only 5% Emulsigen®. The vaccinecaused slight swelling at the site of the injection that resolvedquickly. More significantly, the vaccine induced high concentrations ofborreliacidal OspA and OspC antibodies that peaked a week after thebooster vaccination and remained detectable for the duration of thestudy. In addition, a significant proportion of the OspC borreliacidalantibodies were specific for the highly conserved epitope within OspC7.

B. burgdorferi ss-infected female I. scapularis ticks were then allowedto feed on the vaccinated dogs, and an examination of the midguts fromthe ticks confirmed that the OspA and OspC borreliacidal antibodies inthe bloodmeal completely eliminated B. burgdorferi ss. Specifically, B.burgdorferi ss were detected in 34 (32%) ticks collected from thecontrol dogs vaccinated with placebo and not detected in ticks (n=99)that fed on the dogs vaccinated with the vaccine (p<0.0001). In additionthe vaccine recipients remained negative for Lyme disease by severalindirect and direct methods used commonly to confirm infection. Incontrast, 10 (67%) control dogs produced antibodies against an“infection-specific” 20 kDa B. burgdorferi ss protein and 8 (53%)produced “infection-specific” borreliacidal antibodies. Moreover, four(27%) dogs developed persistent lameness, and B. burgdorferi ss wererecovered from the joints of 3 (75%) of the 4 lame animals. In addition,inflammatory infiltrates developed in the joint capsules of 6 (55%) ofthe 11 placebo-vaccinated dogs examined. More compelling, B. burgdorferiss were recovered from the skin and joints of 14 (93%) and 8 (53)control dogs, respectively, white spirochetes were not recovered fromdogs vaccinated with the test product. Thus, the inventive Lyme diseasevaccine caused minimal side effects and provided complete protectionagainst infection with B. burgdorferi ss.

Example 8 Duration of Immunity of the Vaccine Comprising B. burgdorferiss Isolates S-1-10 and 50772

A. Materials and Methods

Animals:

Materials and methods are described in Example 6 above.

Vaccination and Collection of Sera:

Dogs were vaccinated subcutaneously in the neck with a 1 mL dose of thevaccine and boosted with an additional 1 mL dose after 21 days. Wholeblood was collected prior to the initial (study day −4) and boostervaccination (study day 21) and also on study days 28, 35, 49, 79, 114,142, 175, 210, 238, 266, 302, 322, 357, and 394 by venipuncture of thejugular vein. The serum was separated by centrifugation and stored at−20° C. until tested.

Post-Vaccination Observations:

Dogs were observed daily.

Tick Challenge:

One year after the second vaccination, dogs were shaved on the rightside of the thoracic cavity, and 10 female and 10 male B.burgdorferi-infected I. scapularis ticks were placed in a rubber cupthat was secured to the shaved area with tape and bandage wrap. Theticks were allowed to feed for 7 days.

Detection of B. burgdorferi ss in Ticks:

Detection was performed in the manner described in Example 6 above.

Blood Samples:

Whole blood was collected in serum separation transport (SST) tubes onstudy days 43 post-challenge (PC), 77 PC, 112 PC, 147 PC, 174 PC, and241 PC, and the serum separated and stored at −20° C. until tested.

Detection of Borreliacidal Antibodies:

OspA and OspC borreliacidal antibodies were detected by flow cytometryas described in Examples 1 and 2 above.

Skin Biopsies:

Skin biopsies were taken from sites adjacent to tick attachment on studydays 43 post-challenge (PC), 77 PC, 112 PC, 147 PC, 174 PC, and 241 PCwith a disposable 4 mm puncture device. The biopsies were processed asdescribed in Example 6 above.

Immune Suppression:

Dogs were immune suppressed by daily administration of dexamethasone(0.4 mg/lb body weight) for 5 days beginning at approximately 20 weekspost-challenge.

Clinical Observations:

Clinical observations were performed as in Example 6 above.

Necropsy:

Necropsy was performed as in Example 6 above.

B. Results

Post-Vaccination Serology:

Vaccination with a placebo failed to induce antibodies specific for B.burgdorferi ss. In contrast, vaccination with the vaccine inducedsignificant levels of borreliacidal antibodies detected by using B.burgdorferi ss isolates S-1-10 and 50772. The mean borreliacidalantibody titer against S-1-10 peaked one week (study day 28) after thebooster vaccination, and the response remained detectable until studyday 394 (Table 13). Similarly, high concentrations of borreliacidalantibodies specific for isolate 50772 were detected on study day 28,titers remained elevated on study day 49, and low levels remaineddetectable after 79 days (Table 14).

TABLE 13 Mean titers^(a) (n = 15) of borreliacidal activity^(b) aftervaccination with the vaccine. Day Day Day Day Day Day Day Day Group −421 28 35 49 79 114 142 Vaccinates ND^(c) 192 >7075 >5120 >4457 735 926532 Controls ND ND ND ND ND ND ND ND Day Day Day Day Day Day Day DayGroup 175 210 238 266 302 322 357 394 Vaccinates 702 611 926 557 508 351211 175 Controls ND ND ND ND ND ND ND ND ^(a)Reciprocal dilution.^(b)Detected by using borreliacidal antibody test with B. burgdorferi ssS-1-10. ^(c)ND = None detected. z

TABLE 14 Mean titers^(a) (n = 15) of borreliacidal activity^(b) aftervaccination with the vaccine. Day Day Day Day Day Day Day Day Group −421 28 35 49 79 114 142 Vaccinates ND^(c) 40 1689 532 69 41 ND NDControls ND ND ND ND ND ND ND ND Day Day Day Day Day Day Day Day Group175 210 238 266 302 322 357 394 Vaccinates ND ND ND ND ND ND ND NDControls ND ND ND ND ND ND ND ND ^(a)Reciprocal dilution. ^(b)Detectedby using borreliacidal antibody test with B. burgdorferi ss 50772.^(c)ND = None detected.

Ability of the OspA and OspC Borreliacidal Antibodies to SterilizeInfected Ticks:

Examining the midguts of the ticks that fed on the vaccinated or controldogs confirmed that the OspA and OspC borreliacidal antibodies inducedby the vaccine had sterilized the ticks. B. burgdorferi ss were detectedin the tick smears from 15 (16%) of 95 female ticks that had fed on 12of 15 placebo-vaccinated dogs (Table 15). In contrast, B. burgdorferi sswere detected in the tick smears from only 2 (3%) of 75 female ticksthat had fed on 2 of 15 vaccinated dogs (p=0.0003).

TABLE 15 Detection of B. burgdorferi ss in female ticks removed fromvaccinated or control dogs. Treatment ^(a)Female Ticks Smears PositiveTotal No. of Group Processed for B. burgdorferi Dogs Positive Vaccinates75/148  2/75 2/15 (51%) (3%) Controls 95/146 15/95 12/15* (65%) (16%) *p= 0.0003 ^(a)Engorged and not engorged

Ability of the Vaccine to Prevent Recovery of B. Burgdorferi ss from theSkin:

The vaccine-induced borreliacidal antibodies also prevented a sustainedinfection of B. burgdorferi organisms in the skin. B. burgdorferi sswere recovered from 33 (44%) of 75 biopsies collected from theplacebo-vaccinated dogs at monthly intervals following tick-challenge(Table 16). In contrast, B. burgdorferi ss were recovered from only 6(8%) of 75 biopsies collected from the dogs vaccinated with the vaccine(p<0.0001). Spirochetes were recovered from at least one skin biopsyfrom 10 (67%) of the 15 control dogs, compared to 6 (40%) of the 15vaccinated dogs. However, isolations from the vaccinated dogs werelimited to only the first month post-challenge, whereas 8 (80%) of the10 culture positive control dogs had B. burgdorferi-positive skin biopsycultures throughout the following months of the study.

TABLE 16 Isolation of B. burgdorferi ss from the skin of vaccinated ornon vaccinated control dogs. Day Day Day Day Day Biopsies Dogs DogsTreatment 43 77 112 147 174 Positive Positive Positive Group PC PC PC PCPC (Total)^(b) (Total)^(c) (Total)^(d) Vaccinates 6/15 0/15  0/15  0/150/15 6/75  6/15 0/6 (A) (8%) (40%) (0%) Controls 9/15 8/15* 7/15^(a)*6/15* 3/15  33/75** 10/15  8/10 (B) (44%)  (67%) (80%)  ^(a)One new dog.^(b)Total Biopsies that were positive. ^(c)Total number of Dogs thatwere positive. ^(b)Total number of dogs that were positive after thefirst month. *p < 0.05 **p < 0.0001

Ability of the Vaccine to Prevent Serologic Evidence of Infection:

The vaccine also prevented the development of Lyme disease-specificantibodies. Dogs with Lyme disease produce non-OspC borreliacidalantibodies that are also detectable by using the B. burgdorferi ss 50772isolate (as described in Example 6). Significant, increased levels(≧4-fold increase) of borreliacidal antibodies against strain 50772 wereobserved post-challenge in 5 (33%) of the 15 placebo-vaccinated controldogs compared to none of the dogs vaccinated with the test vaccine.

TABLE 17 Mean titers^(a) of borreliacidal antibodies^(b) after tickchallenge. Total No. of Day Day Day Day Day Dogs with Treatment 43 77112 147 174 Increased Group PC PC PC PC PC Titers Vaccinates 40 40 40 4040 0/15 Controls 48 66 88 88 106  5/15^(c) ^(a)Reciprocal dilution.^(b)Detected by using B. burgdorferi ss 50772. ^(c)≧ 4-fold increasetiter

Ability of the Vaccine to Prevent Frank Limb/Joint Disorders and ErosiveChanges Associated with B. Burgdorferi ss Infection:

Previous studies demonstrated that infection with B. burgdorferi ss onlyrarely causes frank limb/joint disorders (Example 6), and the tickchallenge model has been shown to be less effective in older dogs. Toexacerbate the development of frank symptoms, the dogs wereimmunosuppressed with dexamethasone approximately 3 monthspost-challenge, Four placebo-vaccinated control dogs either became lameor developed erosive lesions in the joint tissue. In contrast, none ofthe dogs vaccinated with a vaccine of the present invention developedany clinical signs of limb/joint disorders or the erosive lesions oftenseen in the joint tissue.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description. Suchmodifications are intended to fall within the scope of the appendedclaims.

It is further to be understood that all base sizes or amino acid sizes,and all molecular weight or molecular mass values, given for nucleicacids or polypeptides are approximate, and are provided for description.

Various publications are cited herein, the disclosures of which arehereby incorporated by reference in their entireties.

We claim:
 1. A vaccine composition comprising an immunologicallyeffective amount of B. burgdorferi ss 50772 (ATCC No. PTA-439) and B.burgdorferi ss S-1-10 (ATCC No. PTA-1680).
 2. The vaccine of claim 1,wherein the immunologically effective amount of B. burgdorferi ss 50772(ATCC No. PTA-439) and B. burgdorferi ss S-1-10 (ATCC No. PTA-1680) havebeen inactivated.
 3. The vaccine composition of claim 2 comprising fromabout 1×10⁴ to about 1×10¹⁰ organisms per milliliter of B. burgdorferiss 50772 and from about 1×10⁴ to about 1×10¹⁰ organisms per milliliterof B. burgdorferi ss S-1-10.
 4. The vaccine composition of claim 3comprising from about 5.0×10⁸ to about 5×10⁹ organisms per milliliter ofB. burgdorferi as 50772 and from about 1.0×10⁸ to about 5×10⁸ organismsper milliliter of B. burgdorferi ss S-1-10.
 5. The vaccine compositionof claim 2 further comprising a pharmaceutically acceptable adjuvant. 6.The vaccine composition of claim 2 further comprising a pharmaceuticallyacceptable immune stimulant.